23-Phenylsteroids

ABSTRACT

Disclosed herein is a novel steroids represented by the formula (1): ##STR1## wherein Z represents ##STR2##  n is integer of 1 to 3, R represents hydrogen, halogen, CF 3 , lower alkyl, lower alkoxy or --CO 2  R&#39;, wherein R&#39; represents hydrogen, alkali metal or lower alkyl, and 
     R 1 , R 2 , R 3  and R 4  represent hydrogen or acyl, respectively. 
     The compound of the formula (1) of the present invention exhibits high durability as well as excellent plant growth regulation.

This application is a division, of application Ser. No. 07/163,526,filed Mar. 3, 1988 now U.S. Pat. No. 4,886,544.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention relates to a novel steroids which is useful as aplant-growth regulator, and to a preparing process thereof.

Brassinosteroids as a plant-growth regulator, were recently remarkedvery much owing to their extremely strong physiological activity. Theireffects vary widely according to the kind and stereoisomerism of theirside chains, so the synthetic research of their derivatives has beenactively conducted.

For example, according to the Japanese Patent Laid-Open No. 227900/1984,26,27-bisnorbrassinosteroid which has not two methyl groups in 26- and27-positions of brassinolide, was found to exhibit the plant-growthpromoting activity as well as the brassinolides.

However, any brassinosteroid compounds substituted by phenyl, whichmight be expected chemically more stable, in their side chain have neverdiscovered in any plant body, nor yet any synthetic examples were known.

The inventors of the present invention have intensively investigated tosynthesize a brassinosteroid derivative which exhibit high durability ofplant growth effect as well as excellent plant growth regulation.

Under these circumstances, it has been found that phenyl which is notyet used, can be successfully introduced in the side chain, and a novelbrassinosteroid represented by the formula (1) mentioned below, can beeasily synthesized. ##STR3## wherein Z represents ##STR4## n is aninteger of 1 to R represents hydrogen, halogen, CF₃, lower alkyl, loweralkoxy or --CO₂ R', wherein R' represents hydrogen, alkali metal orlower alkyl, and

R¹, R², R³ and R⁴ represent hydrogen or acyl, respectively.

In the formula (1), steric configuration of 22-and 23-positions meansone of either (22S,23S)-isomer or (22R,23R)-isomer or a mixture thereof.

The present invention will be illustrated in detail as follows.

A compound of the formula (1) of the present invention can be preparedby the process mentioned hereinafter. Namely, alkenylation (olefinsynthesis, alkene formation) of a compound represented by the formula##STR5## is conducted to obtain a compound represented by the formula:##STR6## wherein R and n are as defined above. As an alkenylation, theremay be mentioned, for example, the following processes.

(1) A compound of the formula (2) is reacted with a compound of theformula: ##STR7## or a compound of the formula: ##STR8## wherein R and nare as defined above,

X represents halogen and

Y represents lower alkyl,

preferably, in molar ratio of 1:1 to 2, in the presence of base in anorganic solvent, preferably at 0° to 40° C.

As organic solvents, aliphatic hydrocarbons such as n-pentane, n-hexaneand heptane, ethers such as diethyl ether, tetrahydrofuran and dioxane,aromatic hydrocarbons such as benzene, toluene and monochlorobenzene,and solvent mixtures thereof may be mentioned. Among them, a solventmixture of benzene-n-hexane is preferable.

Further, as examples of bases, organic lithium compounds such as methyllithium, n-butyl lithium and phenyl lithium, lithium amide ofdialkylamine such as LDA (lithium diisopropyl amine), potassiumt-butoxide and sodium hydride may be mentioned. Among them, n-butyllithium is preferable. Preferable amount used of base is 1 to 1.5 molarratio to 1 mol of the compound of the formula (3) or (3)'. The reactiontemperature is, for example, from -78° C. to the boiling point of thesolvent, preferably near room temperature.

The alkenylation which uses the compound of the formula (3) is known asWittig reaction and the one which uses the compound of the formula (3)'is known as Horner-Emmons reaction [J.I.G. Cadgan "Organic reagent" inOrganic Synthesis, Academic Press, 155, N.Y., (1979)].

(2) The compound of the formula (4) is also obtained by reacting thecompound of the formula (2) with, for example, ##STR9## wherein Mrepresents lower alkyl or phenyl,

R and n are defined as above,

in the presence of alkyl lithium such as C₂ H₅ Li, n--C₃ H₇ Li or n--C₄H₉ Li in an organic solvent such as aliphatic or aromatic hydrocarbon(for example, n-hexane ethylene dichloride, benzene, toluene), ether(for example, diethyl ether, tetrahydrofuran, dioxane),dimethylformaldehyde or HMPA.

A compound represented by the formula (2), as a starting material, maybe obtained by known method, for example, the method similar to thereference example described in Tetrahedron, 38, p. 2099 (1982).

A compound of the formula ##STR10## wherein R and n are as definedabove, which consist of (22S-, 23S)-isomers as main products, can beobtained by oxidizing the compound of the formula (4). The oxidation maybe preferably conducted using catalytic amount of osmium tetroxide inthe presence of a tertiary amine N-oxide such asN-methylmorpholine-N-oxide (NMO), or pyridine in an inert solvent. As aninert solvent there may be mentioned the solvent which are used when thecompound of the formula (4) is produced. The preferable molar ratio is 1to 2 mol of osmium tetroxide per 1 mol of the compound of the formula(4). The oxidation temperature is preferably from room temperature to70° C. In the present invention, method of oxidation is not limited tothe one mentioned above. Any oxidation method that produces cis-glycolmay be used.

Next, the compound of the formula (5) can be hydrolysed preferably byacid catalysts, preferably at room temperature to 100° C. to split offthe protecting group such as ethylene dioxide or acetonide groups, beingconverted to a compound represented by the formula (6). ##STR11##wherein R and n are as defined above.

As examples of the acid catalyst, dilute hydrochloric acid, dilutesulfuric acid, acetic acid or trifluoroacetic acid may be mentioned.Usually, dilute hydrochloric acid or acetic acid is preferable.

Then, a compound of the formula ##STR12## wherein R, R¹, R², R³, R⁴ andn are as defined above, can be obtained by reacting a compound of theabove formula (6) with about 1 to 4 mol of acid anhydride or acidhalide, such as acetic anhydride, trifluoroacetic anhydride, propionicacid anhydride, acetyl chloride, preferably, in the presence of basessuch as pyridine etc., at temperatures preferably from 0° C. to roomtemperature. Subsequently, a lactone compound of the formula ##STR13##wherein R, R¹, R², R³, R⁴ , and n are as defined above, can be derivedby conducting lactonization of a compound of the formula (6) or (7).

As the lactonization method, Baeyer-Villiger reaction may be preferable.Baeyer-Villiger reaction may be performed in similar manner to the usualoxidation reaction using various organic peroxides.

For example, a compound of the formula (6) or (7) may be reacted withorganic peracids such as trifluoroperacetic acid, monoperoxy phthalicacid and m-chloroperbenzoic acid, if necessary, in an organic solventstable to oxidation, such as dichloromethane and perchloroethylene, at alow temperature, preferably -5° C. to 10° C.

As for an organic peracid, the trifluoroperacetic acid prepared in situfrom trifluoroacetic anhydride and hydrogen peroxide may be used mostpreferably.

In addition, a compound of the formula (8) wherein R¹, R², R³ and R⁴ areacyl groups, can be hydrolyzed as usual as in the presence of bases tofree hydroxy groups, respectively.

In the formula (1), as an example of halogen there may be mentioned F,Cl, Br or I, as an example of lower alkyl there may be mentioned methyl,ethyl, propyl, butyl, as an example of lower alkoxy, there may bementioned methoxy, ethoxy or propoxy. As an acyl there may be mentionedacetyl, trifluoroacetyl or propionyl.

The present invention, a result of iterated original investigations, hassucceeded in synthesizing easily a novel plant-growth regulatingsteroidal compound, which has neither been ever isolated from anyplants, nor the existence in plant bodies of which has been ever known,and much less any synthesis thereof, by a very short processes. Saidsteroidal compound may be used to apply to plants for agriculture andhorticulture as a plant growth regulator. For example, it can beutilized to regulate growth of graminaceous crops (cereals) such asrice, wheat and corn, vegetables such as tomato, cabbage, cauliflowerand cucumber, fruit-trees such as grape and apple, beans such as soybean and bush bean, coffee and cocoas.

In accordance with the invention, a steroidal compound of the formula(1) can be used either alone or in the admixture of two or more of saidderivatives by dilution with water in low concentration. Alternatively,they may be mixed with adjuvants to make formulations such as dust,granule, grain, wettable powder, flowable suspension and emulsionconcentrate by means of usual procedures in the agrochemicalmanufacture, to promote or stabilize the effect of said derivatives.

Those adjuvants mentioned above include carriers (diluents) and otheradjuvants such as spreaders, emulsifiers, wetting agents, dispersingagents, fixing agents and disintegrators.

As liquid carriers there can be mentioned aromatic hydrocarbons such astoluene and xylene, alcohols such as methanol, butanol and glycol,ketones such as acetone, amides such as dimethylformamide, sulfoxidessuch as dimethylsulfoxide, methylnaphthalene, cyclohexane, animal andvegetable oils, fatty acids and their esters, etc.

As solid carriers there are mentioned clay, kaolin, talc, diatomaceousearth, silica, calcium carbonate, montmorillonite, bentonite, feldspar,quartz, alumina, sawdust, etc.

As emulsifiers or dispersing agents, surfactants are generally used.They include anionic, cationic, nonionic and amphoteric surfactants suchas sodium higher alkylsulfates, stearyltrimethylammonium chloride,polyoxyethylenealkylphenyl ether, lauryl betaine, etc.

In the case of use of those carriers, it is important to scrutinizecarefully and employ those which are the most suitable for promoting theefficacy of the brassinolide derivatives.

Any of said formulations can be not only alone, but also may be mixedwith fungicides, insecticides, plant growth regulators, acaricides,horticultural pesticides, soil disinfectants, soil improvement agents ornematocides, and further can be used in combination with fertilizers orother herbicides.

The content of a steroidal compound of the formula (1) as activeingredient in the composition varies with types of formulation, methodsof applications and other conditions, and generally it is 0.01 to 95weight %, preferably 0.01 to 50 weight %, though sometimes the activecompound may be used alone.

A plant growth regulating composition of the present invention can beapplied to stem, leaf, seed or root, etc. of said plants, plantcultivation soil or plant cultivation media. In the present inventionthe term "apply" can include "spread", "spray", "sprinkle" or "soak".

It has been remarkably demonstrated that a steroidal compound of theformula (1) in the present invention have plant growth regulating effecteven in very small amounts, and the amount applied is generally 0.00001to 100 mg/are, preferably 0.01 to 10 mg/are. When they are applied as asolution or dispersion in water or an organic solvent, 0.0001 to 10 ppmis preferable.

When applying the composition of the invention for increasing the yieldof cereals, it may be preferable to carry out the applying treatment atabout the time of flowering of said crops. As used herein, the phrase"at about the time of flowering" means the period from the beginning offormation of reproductive cells to the ripening of seeds by completionof fertilization. Taking rice as an example, said period is from thefilling of the ears to the harvest after finishing fertilization of thelast grain flower in the ear.

The present invention will be further illustrated in detail by followingexamples.

SYNTHESIS EXAMPLE SYNTHESIS EXAMPLE 1

A hexane solution of n-butyl lithium (1.6M, 1.9 ml, 3.04 mmol) was addeddropwise to a solution of 1.18 g of benzyltriphenylphosphonium chloridein 10 ml of anhydrous benzene in nitrogen atmosphere at roomtemperature, and stirred for 20 min. Then, a solution of 1.0 g (2.24mmol) of (2R, 3S,20S)-2,3-isopropylidenedioxy-6,6-ethylenedioxy-20-formyl-5α-pregnan in 6ml of benzene was added dropwise thereto, and reacted at roomtemperature for 1 hour. After the reaction mixture was filtered toremove the insoluble precipitate, the organic layer thereof wasseparated by adding water, washed with a saturated aqueous solution ofcommon salt, and dried over anhydrous magnesium sulfate. A crude productobtained by distilling the solvent away under reduced pressure, waspurified by means of a silica-gel column chromatography (eluent:benzene/ethyl acetate) to give 0.86 g (yield 72%) of6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-phenyl-5α,24-norchol-22E-ene(the compound of the formula (4) wherein R=H)

¹ H NMR (CDCl₃), δ0.73 (s, 3H, 18-CH₃), 0.84 (s, 3H, 19-CH₃), 1.11 (d,3H, 21-CH₃), 1.32 (s, 3H, acetonide), 1.47 (s, 3H, acetonide), 3.8 to4.3 (6H, m, ##STR14## 6.01 (dd, J=15.4 and 7.7 Hz, 1H, 23-H), 6.30 (d,J=15.4 Hz, 1H, 22-H), 7.2 to 7.4 (m, 5H, C₆ H₅).

SYNTHESIS EXAMPLE 2

0.69 g (1.3 mmol) of6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-phenyl-5α,24-norchol-22E-enewas dissolved in 4.8 ml of THF, added with 16 ml of t-butanol and 0.8 mlof water, and stirred well. Next, 0.01 g of osmium tetroxide was addedthereto, then also 2.0 g of 60% N-methylmorpholin-N-oxide (NMO), and thereaction was traced by means of TLC. Further, 0.01 g of osmium tetroxideand 4.0 g of 40% NMO were added thereto to accomplish the reaction. 75ml of saturated solution of sodium hydrogen sulfite was added to thereaction mixture, and after stirring for a while the mixture wasfiltered through Celite, extracted by methylene chloride, and theextract was dried over magnesium sulfate. A crude product obtained bydistilling away the solvents under reduced pressure was purified bymeans of silica-gel column chromatography (eluent: n-hexane/ethylacetate) to give 0.28 g (yield 38%) of22,23-dihydroxy-6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-phenyl-5α,24-norcholane(the compound of the formula (5) wherein R=H).

IR (cm⁻¹); 3430 (br, OH)

¹ H NMR (CDCl₃); δ0.64 (s, 3H, 18-CH₃), 0.81 (s, 3H, 19-CH₃), 1.10 (d,3H, 21-CH₃), 1.32 (s, 3H, acetonide), 1.46 (s, 3H, acetonide), 2.46 (d,J=4.9 Hz, 1H), 2.60 (d, J=5.1 Hz, 1H), 3.8 to 4.0 (m, 4H, --OCH₂ CH₂O--), 4.25 (m, 1H), 4.73 (t, J=4.9 Hz, 1H), 7.34 (s, 5H, C₆ H₅).

SYNTHESIS EXAMPLE 3

0.28 g (0.5 mmol) of22,23-dihydroxy-6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-phenyl-5α,24-norcholaneand 12.5 mol of 80% acetic acid were reacted at 50° to 60° C. for 1.5hours, and after cooling, an aqueous solution containing 9.26 g ofsodium carbonate was added thereto. The mixture was extracted four timesby ethyl acetate, and the extract solution was washed with a saturatedaqueous solution of common salt and dried over magnesium sulfate. 0.21 g(yield 89%) of 2α,3α,22,23-tetrahydroxy-23-phenyl-5α,24-norchol-6-one(the compound of the formula (6) wherein R=H) was obtained by distillingaway the solvents under reduced pressure.

IR (cm⁻¹); 3430 (br, OH), 1700 (C═0).

SYNTHESIS EXAMPLE 4

0.21 g (0.45 mmol) of2α,3α,22,23-tetrahydroxy-23-phenyl-5α,24-norchol-6-one was dissolved in3 ml of pyridine, and 0.6 g of acetic anhydride and 0.05 g of4-(N,N-dimethylamino) pyridine were added thereto. After reacted at roomtemperature for 20 hours, the mixture was poured into an ice watercontaining dilute hydrochloric acid. It was extracted three times withethyl acetate, and the extract solution was washed with a saturatedaqueous solution of sodium bicarbonate, and further with a saturatedaqueous solution of common salt, then dried over magnesium sulfate. 0.29g (quantitative yield) of2α,3α,22,23-tetraacetoxy-23-phenyl-5α,24-norchol-6-one (the compound ofthe formula (7) werein R¹ -R⁴ =acetyl and R=hydrogen) was obtained bydistilling away the solvents in reduced pressure.

IR (cm⁻¹); 1780 (COCH₃), 1700 (C═0).

SYNTHESIS EXAMPLE 5

3 ml of methylene chloride solution of trifluoro peracetic acid(prepared from 1.0 g of 35% hydrogen peroxide and 1.6 ml oftrifluoroacetic anhydride at 0° C.) was added to 0.28 g (0.44 mmol) of2α,3α,22,23-tetraacetoxy-23-phenyl-5α,24-norchol-6-one, 1.28 g of Na₂HPO₄ and 15 ml of methylene chloride, and the reaction temperature wasrestored to room temperature. Then the mixture was reacted by boilingunder reflux for 5 hours. By adding an ice water thereto after cooling,methylene chloride layer was separated, and water layer was extractedwell with methylene chloride. Both methylene chloride layer was gatheredtogether, and washed with a saturated aqueous solution of common salt,then dried over magnesium sulfate. 0.27 g of a crude product wasobtained by distilling away the solvent under reduced pressure, and waspurified by means of column chromatography (SiO₂, eluent: n-hexane/ethylacetate=1/5) to give 0.07 g of a mixture of(22R,23R)-2α,3α,22,23-tetraacetoxy-23-phenyl-B-homo-7-oxa-5.alpha.,24-norchol-6-oneand(22S,23S)-2α,3α,22,23-tetraacetoxy-23-phenyl-B-homo-7-oxa-5.alpha.,24-norchol-6-one[IR(cm⁻¹): 1740 (br, CH₃ CO+ ##STR15## ¹ H NMR (CDCl₃); δ 1.988 (s, Ac),2.001 (s, Ac), 2.069 (s, Ac), 2.113 (s, Ac), 2.126 (s, Ac), 2.167 (s,Ac), 7.33 (s, C₆ H₅)] and 0.13 g (yield: 45%) of(22S,23S)-2α,3α,22,23-tetraacetoxy-23-phenyl-B-homo-7-oxa-5.alpha.,24-norchol-6-one[¹ H NMR (CDCl₃); δ 1.991 (s, 3H, Ac), 2.001 (s, 3H, Ac), 2.074 (s, 3H,Ac), 2.116 (s, 3H, Ac), 3.0 (dd, 1H, 5α-H), 4.0 to 4.1 (m, 2H, 7-CH₂),4.88 (m, 1H, 2β-H), 5.17 (dd, 1H, 22 (or 23)-H, 5.37 (br, 1H, 3β-H),6.03 (d, 1H, 23 (or 22)-H), 7.33 (s, 5H, C₆ H₅)].

SYNTHESIS EXAMPLE 6

0.2 g of2α,3α,22,23-tetraacetoxy-23-phenyl-B-homo-7-oxa-5α,24-norchol-6-one wasdissolved in 15 ml of methanol, and 1 ml of aqueous solution of 0.42 gof NaOH was added dropwise thereto, then they were reacted by boilingunder reflux for 3 hours. After cooling, the reaction mixture was addedwith 4.3 ml of 6N-hydrochloric acid and 15 ml of THF, and stirred atroom temperature for 3 hours. After distilling away the solvents underreduced pressure, the residue was neutralized by adding 1.83 g of NaHCO₃powder, and extracted with chloroform three times. After washing by asaturated aqueous solution of common salt, the extract solution wasdried over sodium sulfate, and the solvents were distilled away to give0.134 g (yield 94%) of2α,3α,22,23-tetrahydroxy-23-phenyl-B-homo-7-oxa-5α,24-norchol-6-one (acompound of the formula (8) wherein R═H and R¹ -R⁴ =H), which was amixture of(22S,23S)-2α,3α,22,23-tetrahydroxy-23-phenyl-B-homo-7-oxa-5.alpha.,24-norchol-6-oneand(22R,23R)-2α,3α,22,23-tetrahydroxy-23-phenyl-B-homo-7-oxa-5.alpha.,24-norchol-6-one.

IR and ¹ H NMR showed the following results.

IR (cm⁻¹); 3420 (br, OH), 1710 (CO)

¹ H NMR (CDCl₃ +DMSO-d₆); δ 0.63 (s), 0.64 (s), 0.89 (s), 1.00 (d), 3.1(dd), 3.3 (d), 3.55 to 3.8 (m), 3.95 to 4.1 (m), 4.65 (m), 7.33 (s).

This mixture was purified by means of silica-gel column chromatography(eluent: chloroform/methanol=15/1) to give 28 mg of(22S,23S)-2α,3α,22,23-tetrahydroxy-23-phenyl-B-homo-7-oxa-5.alpha.,24-norchol-6-one,m.p. 146° to 149° C.

This compound showed the following results of IR and ¹ H NMR.

IR (cm⁻¹); 3400 (br, OH), 1715 (C═O).

¹ H NMR (CDCl₃); δ 0.66 (s, 3H, 18-CH₃), 0.88 (s, 3H, 19-CH₃), 1.10 (d,3H, 21-CH₃), 3.08 (dd, 1H, 5α-H), 3.55 to 3.8 (m, 3H), 3.95 to 4.05 (m,2H, 7-CH₃), 4.7 (m, 1H), 7.35 (s, 5H, C₆ H₅).

EI-MS (tetrakistrimethylsilyl compound); m/z 774 (M⁺), 759 (M⁺ -CH₃),668, 595 (base, M⁺ -179, C₂₂ -C₂₃ fission), 505, 463, 415, 375, 357,325, 285, 227, 179, 147. Calcd. for C₄₁ H₇₄ O₆ Si₄ 775, 387.

SYNTHESIS EXAMPLE 7

50 ml of anhydrous benzene was added to a mixture of 8.82 g (3.36×10⁻²mol) of diethyl p-chlorobenzylphosphonate which was obtained by reactingtriethyl phosphite with p-chlorobenzylchloride and 5.00 g (11.2 mmol) of(2R,3S,20s)-2,3-isopropylidenedioxy-6,6-ethylenedioxy-20-formyl-5α-pregnan.1.34 g (3.35×10⁻² mol) of sodium hydride (60%) was added to the abovesolution and allowed to react for three days at room temperature.Another 8.82 g of diethyl p-chlorobenzylphosphonate was added theretoand allowed to react for one day to complete the reaction.

After the reaction, reaction solution was washed with a saturatedaqueous solution of common salt and dried over anhydrous magnesiumsulfate. A crude product obtained by distilling away the solvent underreduced pressure, was purified by means of silica gel columnchromatography (eluent: hexane/ethyl acetate) to give 4.66 g of6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-p-chlorophenyl-5α,24-norchol-22E-ene.

¹ H NMR (CDCl₃); δ 0.72 (s, 3H, 18-CH₃), 0.95 (s, 3H, 19-CH₃), 1.07 (d,3H, 21-CH₃), 1.32 (s, 3H, acetonide), 1.47 (s, 3H, acetonide), 3.8 to4.3 (m, 6H, ##STR16## 6.0 to 6.2 (m, 2H, 22-H, 23-H), 7.23 (s, 4H, C₆H₄)

SYNTHESIS EXAMPLE 8

4.66 g (8.4 mmol) of6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-p-chlorophenyl-5α,24-norchol-22E-enewas dissolved into 42.9 ml of tetrahydrofuran, 142.9 ml of t-butanol and14.3 ml of water were added and stirred well. Then 128 mg of osmiumtetroxide, 3.41 g of N-methyl-morphorine-N-oxide were added thereto andallowed to react for a few days. A saturated solution of sodium hydrogensulfide was added to the reaction mixture and after stirring for awhile, the mixture was filtered through celite, extracted by methylenechloride. After washing with water, the extract was dried over anhydrousmagnesium sulfate. A crude product extract obtained by distilling awaythe solvent under reduced pressure was purified by means of silica gelcolumn chromatography (eluent: n-hexane/ethyl acetate) to obtain 3.40 gof22,23-dihydroxy-6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-p-chlorophenyl-5α,24-norcholane.

¹ H NMR (CDCl₃); δ 0.61 (18-CH₃), 0.66 (18-CH₃), 0.82 (19-CH₃), 1.34(acetonide), 1.46 (acetonide), 3.5 to 4.0 (--OCH₂ CH₂ O--), 4.25 (m),4.68 (m), 7.31 (s, benzene ring proton).

SYNTHESIS EXAMPLE 9

3.30 g (5.6 mmol) of22,23-dihydroxy-6,6-ethylenedioxy-2α,3α-isopropylidenedioxy-23-p-chlorophenyl-5α,24-norcholanewas allowed to react with 60 ml of 80% acetic acid for 2 hours at 50° to60° C. After cooling, the reaction solution was extracted with a mixedsolvent of butanol and ethyl acetate, the obtained extract was washedwith an aqueous solution of sodium carbonate, a saturated aqueoussolution of common salt and dried over anhydrous magnesium sulfate. 2.79g of 2α,3α,22,23-tetrahydroxy-23-p-chlorophenyl-5α,24-norchol-6-one wasobtained after distilling off the solvent under reduced pressure. Theabove compound was a mixture of (22S,23S) and (22R,23R)-tetraol. Themixed compounds were purified by means of silica gel columnchromatography (eluent: chloroform:ethanol:ammonia water=100:3:0.3) toobtain 1.1 g of(22S,23S)-2α,3α,22,23-tetrahydroxy-23-p-chlorophenyl-5α,24-norchol-6-oneand 0.56 g of (22R,23R)-2α,3α,22,23-tetrahydroxy-23-p-chlorphenyl-5α,24-norchol-6-one.

(22S,23S)-isomer

m.p.: 179°-181° C.

¹ H NMR (CDCl₃ +DMSO-d₆); δ 0.65 (18-CH₃), 0.71 (19-CH₃), 3.55 (m), 3.83(dd), 3.97 (d), 4.11 (d), 4.61 (m), 4.75 (d), 7.31 (s, benzene ring).

(22R,23R)-isomer

m.p.: 126°-128° C.

¹ H NMR (CDCl₃ +DMSO-d₆); δ 0.61 (18-CH₃), 0.72 (19-CH₃), 3.4 to 4.0(m), 4.62 (m), 4.71 (d), 7.31 (s, benzene ring).

SYNTHESIS EXAMPLE 10

60 ml of a mixture of acetic anhydride and pyridine (1:9) was added to0.83 g of(22S,23S)-2α,3α,22,23-tetrahydroxy-23-p-chlorophenyl-5α,24-norchol-6-oneand allowed to react for one day at 50° C. After completing thereaction, the reaction mixture was extracted with ethyl acetate, theextract was washed with diluted aqueous solution of hydrochloric acid,saturated aqueous solution of sodium carbonate and saturated aqueoussolution of common salt, dried over anhydrous magnesium sulfate. Thesolvent was distilled off under reduced pressure to give 0.94 g of(22S,23S)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-5α,24-norchol-6-one.

m.p.: 128°-130° C.

¹ H NMR (CDCl₃); δ 0.63 (18-CH₃), 0.81 (19-CH₃), 2.01 (Ac), 2.04 (Ac),2.08 (Ac×2), 4.02 (dd), 4.89 (m), 5.14 (m), 5.28 (m), 6.0 (d), 7.30 (s,benzene ring).

0.52 g of(22R,23R)-2α,3α,22,23-tetrahydroxy-23-p-chlorophenyl-5α,24-norchol-6-onewas acetylated in a similar manner as above to give 0.59 g of(22R,23R)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-5α,24-norchol-6-one.

m.p.: 258°-260° C.

¹ H NMR (CDCl₃); δ 0.64 (18-CH₃), 0.83 (19-CH₃), 2.00 (Ac), 2.02 (Ac),2.08 (Ac), 2.10 (Ac), 4.12 (dd), 4.94 (m), 5.20 (m), 5.38 (m), 6.0 (d),7.30 (s, benzene ring).

SYNTHESIS EXAMPLE 11

Trifluoroperacetic acid was prepared by reacting 0.41 ml of 60% hydrogenperoxide with 2.41 ml of trifluoroacetic anhydride in 10 ml of methylenechloride at 0° C. The whole trifluoroperacetic acid obtained as abovewas added to a solution of 0.705 g (1.0 mmol) of(22S,23S)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-5α,24-norchol-6-one,1.05 g of Na₂ HPO₄ and 50 ml of methylene chloride and allowed to reactat 0° C. Then, the temperature of the reaction mixture was raised toroom temperature and allowed to react under refluxing for 3 hrs. Afterthe reaction, the reaction mixture was washed with an aqueous solutionof common salt, dried over anhydrous magnesium sulfate and the solventwas distilled off under reduced pressure to give 0.733 g of crudeproduct.

The crude product was purified by silica gel column chromatography(eluent: benzen/ethyl acetate) to obtain 559 mg of(22S,23S)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-B-homo-7-oxa-5α,24-norchol-6-one.Similarly, 0.54 g of(22R,23R)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-5α,24-norchol-6-onewas oxidized and resulting crude product was purified to obtain 421 mgof(22R,23R)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-B-homo-7-oxa-5α,24-norchol-6-one.

(22S,23S)-isomer

m.p.: 159°-161° C.

¹ H NMR (CDCl₃); δ 0.66 (18-CH₃), 0.96 (19-CH₃), 1.99 (Ac), 2.00 (Ac),2.07 (Ac), 2.11 (Ac), 3.00 (dd), 4.0 to 4.17 (m), 4.87 (m), 5.12 (m),5.37 (m), 6.00 (d), 7.29, 7.35 (benzene ring).

(22R,23R)-isomer

m.p.: 133°-135° C.

¹ H NMR (CDCl₃); δ 0.67 (18-CH₃), 0.98 (19-CH₃), 2.01 (Ac×2), 2.07 (Ac),2.13 (Ac), 3.00 (dd), 4.05 to 4.15 (m), 4.88 (m), 5.20 (m), 5.38 (m),5.97 (d), 7.30, 7.36 (benzene ring).

SYNTHESIS EXAMPLE 12

430 mg of(22S,23S)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-B-homo-7-oxa-5α,24-norchol-6-onewas dissolved in 30 ml of 1N KOH aqueous methanol solution and allowedto react under reflux for three hours. After cooling, 20 ml of 2Nhydrochloric acid and 50 ml of mixed solvent (butanol-acetic acid wereadded to the above reaction mixture and stirred at room temperature forthree hours. After the usual operation, 300 mg of crude product wasobtained and the crude product was purified by silica gel columnchromatography (eluent: chloroform:ethanol:methanol=100:2:1) to obtain150 mg of(22S,23S)-2α,3α,22,23-tetrahydroxy-23-p-chlorophenyl-B-homo-7-oxa-5α,24-norchol-6-one.Similarly, 102 mg of(22R,23R)-2α,3α,22,23-tetrahydroxy-23-p-chlorophenyl-B-homo-7-oxa-5α,24-norchol-6-onefrom 270 mg of(22R,23R)-2α,3α,22,23-tetraacetoxy-23-p-chlorophenyl-5α-24-norchol-6-one.

(22S,23S)-isomer

¹ H NMR (CDCl₃ +DMSO-d₆); δ 0.67 (s, 3H, 18-CH₃), 0.83 (s, 3H, 19-CH₃),3.10 (dd, 1H, 5.sup.α -H), 3.4-3.65 (m, 3H), 3.8-4.1 (m), 4.15 (m), 4.58(m), 4.81 (d, 1H, 23-H), 7.31 (s, 4H, benzene ring).

m.p.; 213°-214° C.

(22R,23R)-isomer

¹ H NMR (CDCl₃ +DMSO-d₆); δ 0.63 (s, 3H. 18-CH₃), 0.85 (s, 3H, 19-CH₃),3.13 (dd, 1H, 5.sup.α -H), 3.3-3.65 (m), 3.7-4.2 (m), 4.60 (m), 4.80(m), 7.31 (s, 4H, benzene ring).

m.p.; 246°-248° C.

Representative compounds of the present invention, which were preparedaccording to the process for preparation similar to the procedure inabove Synthesis Examples are shown in Table 1.

                                      TABLE 1                                     __________________________________________________________________________     ##STR17##                                                   (1)              Com-                                          IR(cm.sup.-1) or                pound                   Configuration         .sup.1 H NMR (CDCl.sub.3 +                                                    DMSO-d.sub.6)                   No. R.sup.1                                                                            R.sup.2                                                                            R.sup.3                                                                            R.sup.4                                                                            of 22- and 23-                                                                        R     n Z     or m.p.                         __________________________________________________________________________     1  H    H    H    H    (22S,23S)                                                                             H     1                                                                                ##STR18##                                                                          shown in Synthesis Example                                                    6                                2  "    "    "    "    mixture of                                                                            H     1 "     "                                                       (22S,23S) and                                                                 (22R,23R)                                              3  COCH.sub.3                                                                         COCH.sub.3                                                                         COCH.sub.3                                                                         COCH.sub.3                                                                         (22S,23S)                                                                             "     " "     Shown in Synthesis Example                                                    5                                4  "    "    "    "    mixture of                                                                            "     " "     "                                                       (22S,23S) and                                                                 (22R,23R)                                              5  H    H    H    H    mixture of (22S,23S) and (22R,23R)                                                    H     1                                                                                ##STR19##                                                                          shown in Synthesis Example                                                    3                                6  COCH.sub.3                                                                         COCH.sub.3                                                                         COCH.sub.3                                                                         COCH.sub.3                                                                         "       "     "                                                                                ##STR20##                                                                          shown in Synthesis Example                                                    4                                7  H    H    H    H    (22R,23R)                                                                             P-Cl  1                                                                                ##STR21##                                                                          shown in Synthesis Example                                                    12                               8  H    H    H    H    (22S,23S)                                                                             P-Cl  1                                                                                ##STR22##                                                                          shown in Synthesis Example                                                    12                               9  H    H    H    H    (22S,23S)                                                                             P-Cl  1                                                                                ##STR23##                                                                          .sup.1 HNMR δ0.65(s,3H                                                  ,18-CH.sub.3), 0.71(s,3H,19-                                                  CH.sub.3) 2.65(dd,                                                            5α-H), 3.45-3.7(m)                                                      3.83(dd), 4.11(dd) 4.61(m),                                                   4.75(d,1H) 7.31(s, 4H,                                                        benzene ring)                   10  H    H    H    H    (22R,23R)                                                                             m-Cl  1                                                                                ##STR24##                                                                          IR: 3410(br, OH) 1710(CO)                                                     .sup.1 HNMR:δ0.62(S,3H                                                  ,18-CH.sub.3) 0.89(S,3H,                                                      19-CH.sub.3) 3.1(d,d,1H,5.al                                                  pha.-H) 3.6-3.8(m,3H)                                                         3.9-4.1(m,2H) 4.67(m,1H)                                                      7.2-7.4(m,4H, benzene                                                         ring)                           11  "    "    "    "    (22S,23S)                                                                             2,4-di-Cl                                                                           2                                                                                ##STR25##                                                                          IR: 3460(br, OH) 1710(CO)                                                     .sup.1 HNMR:δ0.67(3H,                                                   18-CH.sub.3) 0.93(3H,                                                         19-CH.sub.3) 3.11 (d,d, 1H,                                                   5α-H) 3.6-3.8(m, 3H)                                                    4.07(m, 2H, 7-CH.sub.2)                                                       4.85(m, 1H, 23-H) 7.05(m,                                                     2H, benzene ring) 7.30(m,                                                     1H, benzene ring)               12  "    "    "    "    (22S,23S)                                                                             p-COOH                                                                              1                                                                                ##STR26##                                                                          IR: 3420(br, OH), 2750(br,                                                    OH) 1725(CO), 1705(CO)                                                        .sup. HNMR:δ0.65(3H,                                                    18-CH.sub.3) 0.92(3H,                                                         19-CH.sub.3) 3.20(d,d, 1H,                                                    5α-H) 3.6-3.8(m, 3H)                                                    4.09(m, 2H, 7-CH.sub.2)                                                       4.75(m, 1H ) 7.20(2H,                                                         benzene ring) 7.90(2H,                                                        benzene ring)                   13  H    H    H    H    (22R,23R)                                                                             p-COOCH.sub.3                                                                       1                                                                                ##STR27##                                                                          IR: 3415(br, OH) 1735(CO),                                                    1710(CO) .sup.1 HNMR:δ                                                  0.66(3H, 18-CH.sub.3)                                                         0.95(3H, 19-CH.sub.3)                                                         3.15(d,d, 1H, 5α-H)                                                     3.6-3.8(m, 3H) 3.65(S, 3H,                                                    CO.sub.2 C .sub.--H.sub.3)                                                    4.08(m, 2H, 7-CH.sub.2)                                                       4.75(m, 1H) 7.1(m, benzene                                                    ring)  8.08(m, benzene                                                        ring)                           14  "    "    "    "    (22S,23S)                                                                             m-COOH                                                                              1                                                                                ##STR28##                                                                          IR: 3420(br, OH), 2600(br,                                                    OH) 1730(CO), 1710(CO)                                                        .sup. HNMR:δ0.65(3H,                                                    18-CH.sub.3) 0.95(3H,                                                         19-CH.sub.3) 3.11(d,d, 1H,                                                    5α-H) 3.6-3.8(m, 3H)                                                    4.10(m, 2H, 7-CH.sub.2)                                                       4.85(m, 1H) 7.25(m, 4H,                                                       benzene ring) 7.74(m, 4H,                                                     benzene ring)                   15  "    "    "    "    (22S,23S)                                                                             p-Br  1                                                                                ##STR29##                                                                          .sup.1 H NMR: δ 0.66                                                    (s, 3H, 18-CH.sub.3) 0.74                                                     (s, 3H, 19-CH.sub.3)  2.68                                                    (dd, 1H, 5α-H)                                                          3.6-3.85 (m), 3.95-4.1 (m),                                                   4.65-4.75 (m), 7.23 (d, 2H,                                                   benzene ring), 7.48 (d, 2H,                                                   benzene ring)                   16  H    H    H    H    (22S,23S)                                                                             p-Br  1                                                                                ##STR30##                                                                          m.p.: 222-222.5° C.                                                    .sup.1 H NMR: δ 0.67                                                    (s, 3H, 18-CH.sub.3), 0.86                                                    (S, 3H, 19-CH.sub.3), 3.12                                                    (dd, 1H, 5α-H), 3.4                                                     -3.7 (m), 3.8-4.16 (m),                                                       4.60 (m, 1H), 4.71(d, 1H),                                                    7.26(d,2H, benzene ring),                                                     7.47 (d, 2H, benzene ring)      17  "    "    "    "    mixture of *22S,23S) and (22R,23R)                                                    2,4,5-tri-Cl                                                                        3                                                                                ##STR31##                                                                          IR: 3410(br, OH) 1705(CO)                                                     .sup.1 HNMR:                                                                  δ3.1(d,d,), 3.2(d,d)                                                    (CDCl.sub.3) 4.7-4.85(m)                                                      7.2-7.35(m, benzene ring)       18  "    "    "    "    (22S,23S)                                                                             p-CF.sub.3                                                                          1                                                                                ##STR32##                                                                          IR: 3450(br, OH) 1710(CO)                                                     .sup.1 HNMR:                                                                  δ0.67(3H,                                                               18-CH.sub.3) (CDCl.sub.3)                                                     0.95(3H, 19-CH.sub.3), 3.20                                                   (d,d, 1H, 5α-H)                                                         3.6-3.8(m, 3H) 4.09(m, 2H,                                                    7-CH.sub.2) 4.90(m, 1H)                                                       7.25(d, 2H, benzene ring)                                                     7.48(d, 2H, benzene ring)       19  H    H    H    H    (22S,23S)                                                                             p-CH.sub.3                                                                          1                                                                                ##STR33##                                                                          IR: 3460(br, OH), 1715 (CO)                                                   .sup.1 HNMR: δ0.74                                                      (3H, 18-CH.sub.3) 0.93(3H,                                                    19-CH.sub.3) 2.25(3H, C                                                       .sub.--H.sub.3) 3.12(d,d,                                                     1H, 5α-H), 3.6-3.8(m,                                                   3H) 4.07(m, 2H, 7-CH.sub.2)                                                   .60(m, 1H) 6.8(d, 2H,                                                         benzene ring) 7.05(d, 2H,                                                     benzene ring)                   20  "    "    "    "    (22R,23R)                                                                             p-CH.sub.3                                                                          1                                                                                ##STR34##                                                                          IR: 3450(br, OH)  1715(CO)                                                    .sup.1 HNMR:                                                                  δ0.71(3H,                                                               18-CH.sub.3) (CDCl.sub.3)                                                     0.93(3H, 19-CH.sub.3)                                                         2.30(3H, CH.sub.3) 3.15(d,d,                                                   1H, 5α-H) 3.54(m,                                                      1H) 3.75(m, 1H) 4.1-4.2(m,                                                    3H) 4.65(m, 1H) 7.0(m, 4H,                                                    benzene ring)                   21  "    "    "    "    (22R,23R)                                                                             p-Br  1                                                                                ##STR35##                                                                          .sup.1 H NMR: δ 0.64                                                    (s, 3H, 18-CH.sub.3), 0.85                                                    (s, 3H, 19-CH.sub.3), 3.12                                                    (dd, 1H, 5α-H),                                                         3.3-3.7 (m), 3.8-4.2 (m),                                                     4.58 (m, 1H), 4.82 (d, 1H),                                                   7.25 (d, 2H, benzene ring),                                                   7.45 (d, 2H, benzene ring)                                                    m.p.: 247-249° C.        22  H    H    H    H    (22R, 23R)                                                                            2,4-di-Cl.sub.2                                                                     2                                                                                ##STR36##                                                                          IR: 3460(br,OH)  1710(CO)                                                     .sup.1 HNMR:                                                                  δ0.74(3H,                                                               18-CH.sub.3) 0.95(3H,                                                         19-CH.sub.3) 3.12(d,d, 1H,                                                    5α-H) 3.45(m, 1H)                                                       3.75(m, 1H) 4.0-4.2(m, 3H),                                                   4.90(m, 1H) 7.08(m, 2H,                                                       benzene ring) 7.30(m, 2H,                                                     benzene ring)                   23  "    "    "    "    (22R,23R)                                                                             p-COOH                                                                              1                                                                                ##STR37##                                                                          IR: 3460(br, OH) 2600(br,                                                     OH) 1710(CO), 1705(CO)                                                        .sup. HNMR: δ0.72(3H,                                                   18-CH.sub.3) 0.93(3H,                                                         19-CH.sub.3) 3.11(d.d, 1H,                                                    5α-H) 3.50(m, 1H)                                                       3.77(m, 1H) 4.1-4.2(m, 3H)                                                    4.75(m, 1H) 7.25(d, 2H,                                                       benzene ring) 7.95(d, 2H,                                                     benzene ring)                   24  "    "    "    "    (22R,23R)                                                                             p-COOCH.sub.3                                                                       1                                                                                ##STR38##                                                                          IR: 3450(br, OH), 1730(CO),                                                   705(CO) .sup.1 HNMR:                                                          δ0.75(3H,                                                               18-CH.sub.3) 0.95(3H,                                                         18-CH.sub.3) 3.05(d,d, 1H,                                                    5α-H) 3.41(m, 1H)                                                       3.75(m, 1H) 4.0-4.2(m, 6H,                                                    CO.sub.2 C .sub.--H.sub.3)                                                    4.75(m, 1H) 7.05(d, 2H,                                                       benzene ring)   8.08(d, 2H,                                                   benzene ring)                   25  H    H    H    H    (22R,23R)                                                                             p-Br  1                                                                                ##STR39##                                                                          .sup.1 H NMR: δ 0.61                                                    (s, 3H, 18-CH.sub.3), 0.72                                                    (s, 3H, 19-CH.sub.3), 2.68                                                    (dd, 1H, 5α-H),                                                         3.35-4.0 (m), 4.5-4.7 (m),                                                    7.25 (d, 2H, benzene ring),                                                   .45 (d, 2H, benzene ring)       26  H    H    H    H    (22R,23R)                                                                             p-Cl  1                                                                                ##STR40##                                                                          .sup.1 H NMR: δ 0.61                                                    (s,3H, 18-CH.sub.3), 0.72                                                     (s, 3H, 19-CH.sub.3), 2.68                                                    (dd, 5α-H), 3.35-3.65(                                                  m), 3.8-4.0 (m), 4.65 (dd,                                                    1H,), 4.71(d, 1H) 7.31 (s,                                                    4H, benzene                     __________________________________________________________________________                                                  ring)                       

FORMULATION EXAMPLES FORMULATION EXAMPLE 1 Emulsion

35 parts of a mixture (1:1) of xylene and methylnaphthalene are added toa mixture of 50 parts of Compound No. 1 to form a solution and thesolution is further mixed with 15 parts of a mixture (8:2) ofpolyoxyethylenealkylphenyl ether and calcium alkylbenzenesulfonate toobtain an emulsion.

FORMULATION EXAMPLE 2 Dust

5 parts of Compound No. 1 are mixed with 95 parts of clay and pulverizedto obtain a dust.

FORMULATION EXAMPLE 3 Wettable powder

1 part of Compound No. 8 are mixed with 10 parts of diatomaceous earthand 71 parts of kaolin as the carriers and further uniformly blendedwith 18 parts of a mixture of sodium laurylsulfate and sodium2,2-dinaphthylmethanesulfonate, and thereafter finely pulverized toobtain a wettable powder.

FORMULATION EXAMPLE 4 Granule

0.01 part of a fine dust of Compound No. 7 is spread and coated on 98.4parts of grains (16 to 32 mesh) of silica to obtain a granule, by usinga methanol solution of 1.5 parts of polyvinylacetate as the bindingagent in a proper mixer.

TEST EXAMPLE TEST EXAMPLE 1 Test on lamina inclination of rice plant(lamina joint)

0.5% agar was placed 2 cm deep in a plastic container, 16 cm×16 cm×17cm, and on the surface of the agar, rice seeds, germinated 24 hours ago,(variety: NIHONBARE) were sown, the upper parts of which were covered byaluminium foil. Then, breeding was exercised at 30° C. in the dark for 6days. After that, surroundings of lamina joint parts of the second leafwere cut off about 5 mm across, and the lamina joint parts were floatedin a pure water, and left at 30° C. for 24 hours. Thereafter, 20 samplesof the segment with a uniform angle from every region, were floated inthe laboratory dishes, 6 cm across, containing 5 ml of the processingsolution, and incubated again in the dark at 30° C. for 48 hours. Theangle of the segments was measured and the average for the 20 sampleswas calculated.

    __________________________________________________________________________                                    Degree of                                     Compound                     ppm                                                                              Inclination                                   __________________________________________________________________________     ##STR41##                   0.001 0.01 0.01 1                                                                 51.97 ± 10.5°   83.57 ±                                         10.5°  115.56 ± 10.5°                                        127.83 ± 10.5°                       ##STR42##                   0.001 0.01 0.1 1                                                                  47.60 ± 10.5°  49.66 ±                                          10.5°  92.72 ± 10.5°                                         126.83 ± 10.5°                      __________________________________________________________________________

TEST EXAMPLE 2

Effect of Brassinolide compounds on the growth promotion of sweet corndue to seed dipping treatment

A given amount of sweet corn (variety: Honey bantam 20) were dipped intothe diluted solution of test compounds which were adjusted to eachconcentration for 3 hours under the condition of 25° C.

After that, 6 treated seeds were washed in water, sown into the potwhich packed a given amount of vermiculite and cultivated for 25 days inthe climate chamber which was set the temperature of 15° C. to 20° C.

Plant height and weight of sweet corn were measured 25 days afterseeding. Replication in this trial was carried out 3 times.

    ______________________________________                                        Test results                                                                  Test     Concentration                                                                              Plant height                                                                             Plant weight                                 Compound ppm          %          %                                            ______________________________________                                        Compound 1            114        126                                          No. 1                                                                         Compound 1            114        125                                          No. 2                                                                         Compound 1            111        119                                          No. 5                                                                         Compound 1            115        127                                          No. 7                                                                         Compound 1            114        126                                          No. 8                                                                         Compound 1            116        121                                          No. 12                                                                        Compound 1            115        121                                          No. 16                                                                        Brassinolide                                                                           1            93         111                                          Control  --           100        100                                          ______________________________________                                    

TEST EXAMPLE 3

Effect of Brassinolide compounds on the growth of sweet corn due to seeddipping treatment under the condition of low temperature.

A given amount of sweet corn seeds (variety: Hanny bantam) were dippedinto the diluted solution of test compounds which were adjusted to 0.1and 1 ppm for 3 hours under the condition of 20° C.

After that, 4 treated seeds were washed into water, sown into the potwhich filled up vermiculite and cultivated till they were grown up3-leaf stages in greenhouse. Their sweet corn was exposed the lowtemperature of 5° C. for 5 days.

After that, they were returned to the ordinary temperature of greenhouseagain and cultivated for 8 days there. Plant weight measured 33 daysafter seeding.

    ______________________________________                                        Test results                                                                                 Concentration                                                                             Plant weight                                       Test compound  ppm         %                                                  ______________________________________                                        Compound No. 1 0.1         120                                                               1           116                                                Compound No. 2 0.1         121                                                               1           115                                                Compound No. 8 0.1         122                                                               1           118                                                Compound No. 14                                                                              0.1         120                                                               1           116                                                Compound No. 16                                                                              0.1         123                                                               1           118                                                Brassinolide   0.1         115                                                               1           111                                                Control        --          100                                                ______________________________________                                    

TEST EXAMPLE 4 Effect of Brassinolide compounds on the cell elongationof cucumber

Cucumber seedlings were cultivated in the boxes which packed vermiculitetill a true leaf development.

2 seedlings of cucumber were planted into the pots which contained 100ml diluted solution of test compound which were adjusted to 1 ppm andcultivated for 5 days in the climate chamber under the condition of 20°C. to 25° C.

Petioles of first true leaf of cucumber were cut from the bottom and 1cm segments from their petioles were made. Their segments wereimmediately fixed in FAA solution (Formalin, Acetic acid, Alcohol) andenclosed by paraffin.

Segments for preparats were made from their materials by Microtome. Cellsize of their segments was measured by Microscope.

    ______________________________________                                        Test results                                                                  Test          Concentration                                                                             Cell size of                                        Compound      (ppm)       cucumber (mμ)                                    ______________________________________                                        Compound No. 1                                                                              1           135.1                                               Compound No. 8                                                                              1           136.2                                               Compound No. 15                                                                               0.5       129.0                                               Compound No. 21                                                                             1           131.5                                               Gibberellin   1           120.3                                               IAA           1           105.5                                               Control       --          110.4                                               ______________________________________                                    

What we claim is:
 1. A steroidal compound represented by the formula:##STR43## wherein Z represents ##STR44## n represents an integer of 1 to3, R represents hydrogen, halogen, CF₃, lower alkyl, lower alkoxy or--CO₂ R', wherein R' represents hydrogen, alkali metal or lower alkyl,andR¹, R², R³ and R⁴ represent hydrogen or acyl, respectively,with theproviso that when n is 2 or 3, R may represent substituents which may beindependent each from one or more of the others.
 2. A steroidal compoundaccording to claim 1, wherein Z represents ##STR45## n represents aninteger of 1 to 3, R represents hydrogen, halogen, CF₃, lower alkyl, or--CO₂ R' wherein R' represents hydrogen, alkali metal or lower alkyl,and R¹, R², R³ and R⁴ represent hydrogen or acetyl, respectively, withthe proviso that when n is 2 or 3, R may represent substituents whichmay be independent each from one or more of the others.
 3. The steroidalcompound according to claim 2 of the formula: ##STR46## wherein R⁵ isselected from the group consisting of ##STR47##
 4. A plant growthregulating composition comprising an effective plant growth regulatingamount of a steroidal compound of the formula: ##STR48## wherein Zrepresents ##STR49## n represents an integer of 1 to 3, R representshydrogen, halogen, CF₃, lower alkyl, lower alkoxy or --CO₂ R', whereinR' represents hydrogen, alkali metal or lower alkyl, andR¹, R², R³ andR⁴ represents hydrogen or acyl, respectively,with the proviso that whenn is 2 or 3, R may represent substituents which may be independent eachfrom one or more of the others, and one or more acceptable adjuvants. 5.A method for regulating plant growth which comprises applying aneffective plant growth regulating amount of a compound of the formula:##STR50## wherein Z represents ##STR51## n represents an integer of 1 to3, R represents hydrogen, halogen, CF₃, lower alkyl, lower alkoxy or--CO₂ R', wherein R' represents hydrogen, alkali metal or lower alkyl,andR¹, R², R³ and R⁴ represent hydrogen or acyl, respectively,with theproviso that when n is 2 or 3, R may represent substituents which may beindependent each from one or more of the others, to said plant and locusthereof.